转Cry1Ab基因水稻分子特征及其特异性PCR检测方法

doi:10.3724/SP.J.1005.2012.00208

遗传 ›› 2012, Vol. 34 ›› Issue (2): 208-214.doi: 10.3724/SP.J.1005.2012.00208

转Cry1Ab基因水稻分子特征及其特异性PCR检测方法

汪小福¹, 陈笑芸¹, 张小明², 周育¹, 张焕春³, 缪青梅¹, 方敬¹, 徐俊锋¹

1. 浙江省农业科学院农产品质量标准研究所，杭州 310021 2. 浙江省农业科学院作物与核利用研究所，杭州 310021 3. 沈阳师范大学化学与生命科学学院，沈阳 110034

收稿日期:2011-05-24 修回日期:2011-06-29 出版日期:2012-02-20 发布日期:2012-02-25
通讯作者: 徐俊锋 E-mail:njjfxu@yahoo.com.cn
基金资助:
浙江省重大项目(编号：2008C12074)和浙江省自然科学基金项目(编号：Y3090252, Y3090577)资助

Molecular characteristics and specific PCR detection of transgenic rice containing Cry1Ab

WANG Xiao-Fu¹, CHEN Xiao-Yun¹, ZHANG Xiao-Ming², ZHOU Yu¹, ZHANG Huan-Chun³, MIAO Qing-Mei¹, FANG Jing¹, XU Jun-Feng¹

1. Institute of Agricultural Quality and Standard, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China 2. Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China 3. College of Chemistry and Life Science, ShenYang Normal University, Shenyang 110034, China

Received:2011-05-24 Revised:2011-06-29 Online:2012-02-20 Published:2012-02-25

摘要/Abstract

摘要： 转Cry1Ab基因水稻Bt01为一种新型的转基因水稻, 文章首先利用Southern blotting验证了外源基因Cry1Ab转入了Bt01中, 且为单拷贝, 再利用TAIL-PCR方法获得了其插入位点信息, 根据获得的Bt01的5′端插入位点序列, 设计了相应的定性与定量PCR检测体系的引物及探针, 实验结果显示, 定性PCR检测体系的最低检测极限(LOD)为10个拷贝, 定量PCR检测体系的LOD为5拷贝, 最低定量极限(LOQ)为10拷贝。同时为了验证建立的定量PCR体系的准确性, 利用该体系检测已知转基因水稻Bt01含量分别为3%和0.5%的样品, 定量结果分别为2.7%和0.47%。研究结果表明, 该转化体特异性定性与定量检测方法具有高度的特异性和良好的灵敏性, 为转基因水稻Bt01的身份识别和检测提供了有效的方法。

关键词: 分子特征, 转基因, Cry1Ab, 定性与定量PCR, 特异性检测

Abstract: Bt01 is a new type of rice that has been genetically modified to express Cry1Ab protein. This study confirmed that Cry1Ab was inserted into Bt01 as a single copy using Southern blotting analysis. TAIL-PCR method was further used to obtain its insertion siteinformation. Specific PCR primers and TaqMan probes were designed based on the 5′-integration junction sequence of transgenic rice Bt01. The results showed that the limit of detection (LOD) was ten copies in qualitative PCR. The quantitative PCR assay showed that the LOD was five copies, and the limit of quantification (LOQ) was ten copies. In addition, the accuracy of the established quantitative PCR was verified by detecting two samples containing 3% and 0.5%Bt01, respectively. The quantitative PCRanalysis showed the results were 2.7% and 0.47%, respectively.The above results indicated that the event-specific PCR methodsdeveloped have high specificity and good sen-sitivity, which could be effective methods for identifying and testing the genetically modified Bt01 rice.

Key words: molecular characteristics, transgenic, Cry1Ab, qualitative and quantitative PCR, specific detection

汪小福，陈笑芸，张小明，周育，张焕春，缪青梅，方敬，徐俊锋. 转Cry1Ab基因水稻分子特征及其特异性PCR检测方法[J]. 遗传, 2012, 34(2): 208-214.

WANG Xiao-Fu, CHEN Xiao-Yun, ZHANG Xiao-Ming, ZHOU Yu, ZHANG Huan-Chun, MIAO Qing-Mei, FANG Jing, XU Jun-Feng. Molecular characteristics and specific PCR detection of transgenic rice containing Cry1Ab[J]. HEREDITAS, 2012, 34(2): 208-214.

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转Cry1Ab基因水稻分子特征及其特异性PCR检测方法

Molecular characteristics and specific PCR detection of transgenic rice containing Cry1Ab

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